FIG. 2.

Evidence that the EphA8 JM domain associates directly with the PTB domain of Anks family proteins. (A) Demonstration of AIDA-1 PTB domain interaction with the EphA8 JM domain, using purified proteins. Purified GST or GST-JM fusion proteins were mixed with purified His-tagged AIDA-1 PTB protein. Bound proteins were precipitated using His-bind resin, fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and detected by Western blotting with anti-GST antibody (top panel). The same blot was stripped and reprobed with anti-AIDA antibody (middle panel). GST or GST-JM fusion proteins used for this experiment were directly detected by Coomassie staining (bottom panel). (B) Demonstration of AIDA-1 PTB domain interaction with the full-length EphA8 receptor. Purified proteins (GST or GST-PTB) were mixed with whole-cell lysates from HEK293 cells transfected with the control vector (lanes 1 and 2) or an EphA8 expression construct (lanes 3 and 4). Bound proteins were pulled down using glutathione beads, fractionated by 10% SDS-PAGE, and detected by Western blotting using anti-EphA8 antibody (top panel). The same blot was stripped and reprobed with anti-GST antibody (middle panel). A sample (2%) of each total cell lysate was analyzed directly by Western blotting using anti-EphA8 antibody (bottom panel). (C) Evidence that the Odin PTB domain interacts with the EphA8 JM domain, using purified proteins. Experiments were performed essentially as described for panel A, except that purified His-tagged Odin PTB protein and anti-Odin antibody were used. PD, pulldown; WB, Western blot.